This invention relates to a laser illumination and detection system for performing sample analysis, such as DNA sequencing, DNA fingerprinting, absorption/emission spectroscopy, and the like. More particularly, it pertains to a laser illumination system that employs a laser scanner.
A conventional capillary array electrophoresis system is configured to perform a high-throughput analysis on biological samples, e.g., DNA sequencing, using a highly sensitive laser-induced fluorescence detection method. In particular, the samples to be analyzed either possess fluorescing functional groups (fluorophores) in their molecular structure or are tagged with fluorescent dyes. These samples are then excited with a laser beam which causes the samples to emit fluorescence light. The emitted fluorescence light is detected and subsequently analyzed.
The samples are illuminated by the laser beam either while they are still migrating through the capillaries, i.e., on-column detection, or after they elute from output ends of the capillaries, i.e., sheath-flow detection, as described by Dovichi et al. (U.S. Pat. No. 5,741,412).
For the on-column detection method, samples in the confocal microscope scanning method can be used as described in Mathies el al. (U.S. Pat. No. 5,274,240) In this method, samples in each capillary are sequentially excited and detected by a confocal scanning system. In another method, as described by Yeung et al,(U.S. Pat. No. 5,741,411) all the capillaries are illuminated by a laser beam and monitored by a 2-dimensional charged couple device (CCD) simultaneously.
FIG. 1 illustrates a conventional on-column detection system 1 that includes a laser light source 3 illuminating a capillary array 5 and samples therein and a camera lens 7 receiving the emitted light from the samples. Subsequently, the received light from the samples is captured by a CCD and analyzed.
FIG. 2 shows an intensity profile, amounts of light received across a viewing field of the camera 7. More specifically, the measurements are made by illuminating a laser beam on the array of capillaries 5 having the same quantity of samples migrating through each of the capillaries. The view field of the camera 7 is about 2 cm, i.e., the width of a 96 capillary array comprising capillaries with 200 xcexcm outside diameters (o.d.)laid side by side. The position of 300 in FIG. 2 corresponds to the center of the array.
The resulting intensity profile shows that the amount of the light received from the location near the center of the array is more than that from capillaries at the periphery of the array.
At least two aspects of the conventional system 1 cause this effect. First, the laser beam has a Gaussian beam profile. In other words, a laser beam produced by a conventional laser illuminates the capillaries in the middle portion with about 1.5 times more intensity than the capillaries at the periphery of the array. Second, the amount of light captured by the camera varies based on the location of the capillaries. In particular, the amount of light received by the camera from a unit area of the capillaries at the periphery of the array is less than that from a unit area of the capillary at the center of the array, when an identical amount of light is emitted by the samples within the capillaries in each of the unit areas.
The above discussed shortcomings of the conventional system produce a non-uniform intensity profile. For instance, the amounts of light received from the center capillaries and periphery capillaries can differ by a factor of 2-4, as shown in FIG. 2.
The non-uniform intensity profile is not desirable, because in order to obtain sufficient amounts of light from the capillaries at the periphery of the array, the strength of the laser beam illuminating the center of the array may saturate the camera. Further, in order to process and analyze the data collected under this condition for capillary-to-capillary comparison and quantification, the subsequent analysis. process becomes complicated.
The present invention, therefore, provides a capillary electrophoresis system that includes capillaries positioned parallel to each other to form a plane. The capillaries are configured to allow samples to migrate therethrough. The system further includes a light source configured to illuminate the capillaries and the samples therein. This causes the samples to emit light. The system also includes a lens configured to receive the light emitted by the sample. The lens is positioned directly over a first group of the capillaries and obliquely over a second group of the capillaries. The light source is further configured to illuminate the second group of capillaries more than the first group of the capillaries such that amount of light received by the lens from the first group of capillaries is substantially identical to amount of light received from the second group of capillaries when an identical amount of the samples is migrating through the first and second group capillaries.
The light source further includes a laser configured to produce a laser beam and a scanning mirror optically coupled to the laser to receive the laser beam. The scanning mirror is configured to be oscillated and positioned to aim the received laser beam at the capillaries. The light source further includes a control device operatively coupled to the scanning mirror. The control device is configured to control the oscillation of the scanning mirror. This causes the laser beam from the scanning mirror to illuminate the plurality of capillaries.
The plane formed by the plurality of capillaries has a coincident axis extending parallel to the lengths of the capillaries. Further, the scanning mirror aims the laser beam through a scanning plane which is formed by a locus of the laser beam illuminating the capillaries. In turn, the laser beam impinges on the capillaries at an angle of 45xc2x0-90xc2x0 formed between the scanning plane and the coincident axis. The plane formed by the plurality of capillaries also has a transverse axis extending parallel to the widths of the capillaries. Further, the scanning plane has a central beam line extending from the scanning mirror to a center point among the capillaries illuminated by the laser beam. The laser beam impinges on the capillaries at an angle of 1xc2x0-90xc2x0 formed between the transverse axis and the central beam line.
The present invention also provides a capillary electrophoresis method that includes the steps of introducing samples to a plurality of capillaries positioned in parallel to each other forming a plane and forming a first group and a second group of capillaries, and causing the samples to migrate through the capillaries. The method also includes the step of illuminating the second group of capillaries more than the first group of the capillaries such that amount of light received by a lens from the first group of capillaries is substantially identical to amount of light received from the second group of capillaries when an identical amount of the samples is migrating through the first and second group capillaries. The lens is positioned directly above the first group of capillaries and obliquely over the second group of capillaries.
In one embodiment, the method also includes the steps of measuring amount of light received by the lens from the first and second groups of capillaries, while injecting an identical amount of the samples into the first and second capillaries, and while illuminating the first and second groups of capillaries with a substantially identical amount of light. Subsequently, a difference between the amount of light received by the lens from the first and second groups of capillaries is calculated.
Furthermore, the illuminating step further includes the steps of generating a compensating laser beam that substantially eliminates the calculated difference. The capillaries are illuminated by the compensating laser beam.